The role of surface receptors [Toll like receptors (TLRs) and Fc gamma receptors (Fc3R)] in susceptibility to pediatric severe malarial anemia Severe malarial anemia (SMA) is the most common clinical manifestation of severe malaria in infants and young children living in holoendemic Plasmodium falciparum transmission areas. This particular clinical manifestation accounts for the greatest amount of global malaria-related morbidity and mortality. The pathophysiology of SMA is multi-factorial and has been attributed to direct destruction of red blood cells (RBCs) by parasites, hemolysis of non-parasitized RBCs, suppression of erythropoiesis, erythrophagocytosis, and dysregulation in cytokine production. However, the current lack of knowledge about the genetic and immunological basis of SMA poses a significant problem since a successful malaria vaccine must protect against the development of severe life- threatening SMA. The Toll-like receptors (TLRs) have been recognized as key regulators of the immune response. TLRs are crucial components of the innate immune response to various microbial ligands and reactive host products. TLR ligation results in generation of inflammatory sequelae that links the generalized innate immune response with more specific immunological systems (adaptive immunity). Recently, three TLRs have been identified that recognize Plasmodium falciparum: TLR-2 and TLR-4 that respond to the putative toxin glycosylphosphatidylinositol (GPI);and TLR-9 that responds to malarial pigment (hemozoin, Hz). Previous studies demonstrated that frequent polymorphisms within TLR-4, but not TLR-9, confer an increased risk of severe pediatric malaria characterized by mixed sequelae of cerebral malaria (CM) and/or SMA. Another recent study also showed that TLR-4 and TLR-9 variants play a role in the manifestation of malaria during pregnancy. Since TLRs affect the innate and adaptive immune responses, the role of polymorphic variants within the TLR-4 and TLR-9 in conditioning susceptibility to SMA will be investigated in the current proposal. In addition, this proposal will determine the impact of polymorphic variants within the TLRs on interferon (IFN)-3 production, an important immunological mediator for both the innate and adaptive immune responses. This follows previous observations that demonstrated that IFN-3 may be playing dual roles in malaria pathogenesis;serving as an essential component of host defense, while also contributing to enhanced pathogenesis. For example, studies in experimental murine malaria have shown that IFN-3 exhibits anti- parasitic effects, yet it is also involved in promoting neurological complications and profound malarial anemia. In addition, cells from children that produce high levels of IFN-3 after in vitro challenge with P. falciparum antigen are more likely to experience severe clinical manifestations of malaria. Fc3 receptors (Fc3R) acts as a crucial link between the humoral and cellular immune responses. The major function of Fc3Rs is activation of accessory cells against pathogens, making these receptors essential for host defense against infection. Previous studies carried out in low malaria endemic areas demonstrated that Fc3RIIa- 131H/H genotype, in combination with Fc3RIIIb-NA2 allele, is associated with susceptibility to CM, while a recent study found no association between the Fc3RIIIa-176F/V polymorphism with malaria disease outcomes in the same population. Studies carried out in Kenyan infants demonstrated that the Fc3RIIa-131R/R genotype was associated with protection against both a lower and higher threshold of high-density parasitemia (HDP;e10,000 parasites/5L). The Fc3RIIIa gene is located proximate to Fc3RIIa and Fc3RIIIb, and the three genes have been shown to be in linkage disequilibrium. A recent report on a dimorphism within the Fc3RIIIa at position 176 (F/V) demonstrated that variation at this location influences the binding of IgG1, IgG3 and IgG4. In P. falciparum malaria, IgG1 and IgG3 are associated with lower parasitemia and reduced risk of infection. As such, it is important to carry out further investigations that examine both single nucleotide polymorphisms (SNPs) and haplotypic structures within the Fc3R gene and their respective association with SMA in holoendemic P. falciparum transmission areas, such as western Kenya. The overall objective of this proposal, therefore, is to investigate the functional impact of variation within these receptors (TLR and Fc3R genes) in regulating the development and clinical outcomes of SMA in children less than 3 years of age in a holoendemic P. falciparum transmission area of western Kenya. The overall hypothesis of this proposal is that these critical receptors play a significant role in the immunopathogenesis of severe malaria and, hence, condition the clinical outcomes of childhood malaria. To accomplish the experimental objectives, we will utilize a sample repository, as well as prospectively collected samples acquired as part of our ongoing investigations. Since the underlying molecular basis required for development of protective immunity against SMA still remain largely undefined, our investigations will focus on important receptors that link the innate and adaptive immune systems. The results from these investigations in phenotypically well-defined cohorts of children will maximize our ability to successfully identify genotypes/haplotypes that condition susceptibility to SMA. This information will be important since the primary goal of our genetic-based investigations is to identify susceptible groups for targeted therapeutic interventions. PUBLIC HEALTH RELEVANCE: Severe malarial anemia (SMA) is the most common clinical manifestation of severe malaria in infants and young children living in holoendemic Plasmodium falciparum transmission areas such as western Kenya. This clinical manifestation accounts for the greatest amount of worldwide malaria-related morbidity and mortality. The pathophysiology of SMA is multi-factorial and has been attributed to direct destruction of red blood cells (RBC) by parasites, hemolysis of non-parasitized RBC, suppression of erythropoiesis, erythrophagocytosis, and imbalances in cytokine production. However, the current lack of knowledge about the genetic and immunological basis of SMA poses a significant problem since a successful malaria vaccine must protect against the development of severe life-threatening SMA. To address this issue, we have focused on accurately phenotyping the clinical features that define SMA so that genetic-based investigations can identify critical immunological pathways that mediate disease outcomes. Accomplishment of this task that takes into account the genetic, inflammatory, and clinical factors that promote SMA will be useful for predicting disease outcomes. In the region for the proposed investigations, perennial P. falciparum is responsible for ~97% of the malarial infections, resulting in ~20% mortality rate in children less than 36 mos. of age. Since our previous studies have shown that dysregulation in immune mediators is associated with malaria disease severity, the current investigations will focus on the impacts of surface receptors [Toll like receptors (TLRs) and Fc gamma receptor (Fc3R) genes on pediatric SMA. Since the underlying molecular basis required for development of protective immunity against SMA still remain largely undefined, our investigations will focus on important receptors that link the innate and adaptive immune systems. Carrying out these investigations in phenotypically well-defined cohorts of children will maximize our ability to successfully identify genotypes/haplotypes that condition susceptibility to SMA.